The central objective of this proposal is to identify the design principles by which gene clusters encoding complex molecular machines are organized and regulated. As a model system, we will study the type III secretion system (T3SS) encoded in Salmonella Pathogenicity Island 1 (SPI-1). The T3SS is a molecular machine that acts a syringe to inject proteins into host cells. The T3SS requires the expression of ~18 proteins at the correct ratios to properly assemble and function. Little is known as to how changes in the relative expression levels, the order of expression, and expression noise impact this process. We propose to study SPI-1 by rebuilding it from the bottom-up, using biophysical models and well-characterized genetic parts. This process, known as refactoring, replaces all of the regulation with synthetic variants and eliminates unknown and poorly characterized regulation. The refactored SPI-1 is controlled by synthetic genetic circuits that implement the dynamics of gene expression. In Aim 1, the refactored system will be used as a platform to determine how gene order and translational coupling impact the robustness of the cluster to perturbations in expression levels. In Aim 2, the impact of different regulatory programs on the proper assembly and function of the T3SS will be determined. Synthetic genetic circuits will be constructed that implement different feed forward and feedback loops that are predicted by mathematical models to affect the noise and dynamics of gene expression. This enables the testing of hypotheses that controlling the temporal order and intrinsic noise of expression is important for the assembly of macromolecular complexes. Together, these studies will identify robust and fragile aspects of common virulence mechanism shared by many human pathogens.